Antiprotozoal activity of different Xenorhabdus and Photorhabdus bacterial secondary metabolites and identification of bioactive compounds using the easyPACId approach

Natural products have been proven to be important starting points for the development of new drugs. Bacteria in the genera Photorhabdus and Xenorhabdus produce antimicrobial compounds as secondary metabolites to compete with other organisms. Our study is the first comprehensive study screening the anti-protozoal activity of supernatants containing secondary metabolites produced by 5 Photorhabdus and 22 Xenorhabdus species against human parasitic protozoa, Acanthamoeba castellanii, Entamoeba histolytica, Trichomonas vaginalis, Leishmania tropica and Trypanosoma cruzi, and the identification of novel bioactive antiprotozoal compounds using the easyPACId approach (easy Promoter Activated Compound Identification) method. Though not in all species, both bacterial genera produce antiprotozoal compounds effective on human pathogenic protozoa. The promoter exchange mutants revealed that antiprotozoal bioactive compounds produced by Xenorhabdus bacteria were fabclavines, xenocoumacins, xenorhabdins and PAX peptides. Among the bacteria assessed, only P. namnaoensis appears to have acquired amoebicidal property which is effective on E. histolytica trophozoites. These discovered antiprotozoal compounds might serve as starting points for the development of alternative and novel pharmaceutical agents against human parasitic protozoa in the future.

Since protozoan parasites are eukaryotic organisms that share functional homology with mammalian cells, currently available drugs for the treatments of parasitic diseases are generally toxic to human cells and have adverse side effects 7,8 . Owing to these undesired effects and considering the development of resistant strains of parasites against pharmaceutical products, new drugs with different modes of action on target parasites and minimal toxicity to host cells are urgently required 9,10 .
Natural products (or secondary metabolites) have been proven to be an important starting point for the development of new drugs. Screening natural products provide the chance of discovering new molecules with unique structure, high activity, and selectivity 11 . The most important natural product sources in nature are fungi 12,13 , plants 14 and bacteria 11,15,16 . Various fungi and bacteria produce antimicrobial compounds as secondary metabolites to compete with other organisms. One of the sources of novel bioactive therapeutics against parasites are insect pathogenic Photorhabdus and Xenorhabdus bacteria. These bacteria encode several putative biosynthetic pathways for natural product biosynthesis [17][18][19] of which several of them are conserved since they fulfill important ecological functions in their ecological niche 20 . Photorhabdus and Xenorhabdus bacteria are associated with entomopathogenic nematodes which are obligate and lethal insect parasitic organisms 21,22 . When these nematodes penetrate an insect host, they release their mutualistic bacteria into the insect hemolymph and within 48 h the insect host is killed because of bacterial toxins and enzymes 23,24 . Furthermore, to protect the nematode-infected cadaver from opportunistic microorganisms (e.g., bacteria, fungi, protozoa, and viruses) both Xenorhabdus and Photorhabdus bacteria produce a variety of natural products that have antimicrobial activities 19,25,26 . Although several studies have reported the antibacterial [27][28][29][30] , antifungal [29][30][31][32][33][34][35] , and insecticidal [36][37][38] activities, only very few studies have investigated the antiprotozoal effect of the secondary metabolites produced by these bacteria 39,40 . Currently, more than 40 different species of Photorhabdus and Xenorhabdus bacteria have been identified 23,41 that produce different sets of natural products 42 . The aim of our study was to investigate natural products produced by five Photorhabdus and 22 Xenorhabdus species against human parasitic protozoa, A. castellanii, E. histolytica, T. vaginalis, L. tropica, and Trypanosoma cruzi (T. cruzi), and the identification of novel bioactive antiprotozoal compounds by using the easyPACId (easy Promoter Activated Compound Identification) approach 43 .

Material and methods
Bacterial sources and preparation of cell-free supernatants. The cell-free supernatants of 22 Xenorhabdus and 5 Photorhabdus species were tested against human parasitic protozoa (Table 1). All bacteria strains were obtained from the Bode lab and were kept at − 80 °C as stock culture until use.
A loopfull of bacteria taken from stock culture was inoculated to Luria Bertani (LB) (Merck) agar medium and incubated at 30 °C for 24 h. A single colony was picked and inoculated to 10 ml sterilized Tryptic Soy Broth (TSB) medium (Merck) and cultivated at 30 °C for 24 h to be used as overnight culture. Subsequently, 1 ml from overnight culture was transferred to 50 ml sterilized TSB medium and incubated at 30 °C and 150 rpm for 120 h (it is known that these bacteria produce the most secondary metabolite after 120 h) 30,44 . To obtain cell-free supernatant, the bacterial broth was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was collected carefully and filtered through a 0.22 μm Millipore filter (ISOLAB) 45 . An aliquot of the filtrated suspension was streaked onto NBTA agar to verify the absence of bacterial cells 46 . The supernatants were poured into the 50 ml sterile centrifuge tubes (Corning, NY) and kept at − 20 °C for up to 2 weeks prior to use 47,48 . In vitro cultures of parasitic protozoons. Axenic cultures of A. castellanii trophozoites (ATCC 30010) were maintained in liquid PYG (protease peptone-yeast extract-glucose) medium supplemented with penicillin G (500 U/ml) and streptomycin (50 μg/ml) 49 (Pérez-Serrano et al. 2000). The cultures were refreshed weekly in 25 ml cell culture flasks (Sigma) and incubated at 30 °C, until use 50,51 . Cells from the culture medium were harvested by centrifugation at 2000 rpm for five minutes and washed three times with Phosphate-Buffered Saline (PBS). Acanthamoeba castellanii trophozoites adhering to flasks were collected by placing the flasks on ice for 30 min with gentle agitation 52,53 .
Entamoeba histolytica (ATCC 30459) strain was kindly provided by Dr. Charles Graham Clark from the London School of Hygiene and Tropical Medicine. Entamoeba histolytica trophozoites were cultured axenically in LYI medium (880.0 ml LYI Broth, 20.0 ml Vitamin Mixture, 100.0 ml Heat Inactivated Adult Bovine Serum) supplemented with penicillin G (500 U/ml) and streptomycin (50 μg/ml) 54 . The cultures were routinely maintained by subculturing into screw capped test tube containing 7 mL of LYI medium 55,56 .
Trichomonas vaginalis (ATCC 30001) trophozoites were grown in Diamond's trypticase yeast-extract maltose (TYM) medium (0.5 mg of L-cysteine HCl, 0.1 g of ascorbic acid, 0.4 g of K 2 HPO 4 , 0.4 g of KH 2 PO 4 , 10 g of trypticase, 2.5 g of maltose and 10 g of yeast extract in one ml of distilled water, pH:6) supplemented with 100 IU/ml streptomycin, 100 IU/ml penicillin and 10% heat-inactivated Fetal Bovine Serum (FBS). T. vaginalis subcultures were cultured regulaly to maintain viability and for use in the assays 57 .
Trypanosoma cruzi (CBU-TC01) trypomastigotes were obtained from the parasite biobank of Manisa Celal Bayar University School of Medicine Department of Parasitology Manisa, Turkey. The trypomastigotes were incubated at 27 °C in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 200 U penicillin/ ml, and 0.2 mg streptomycin/ml. Subcultures were maintained in 25 ml flasks until use in the experiments 59 Two methods were used to determine the antiprotozoal effects of the bacterial supernatants in vitro. To assess the anti-leishmanial activity was performed by using the XTT (sodium 3,39-[1-(phenylaminocarbonyl)-3,4tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) cell proliferation kit (Roche Molecular Biochemicals, Mannheim, Germany) as previously described 63 .
Cell viability assay test was used for A. castellanii, T. vaginalis, E. histolytica and T. cruzi. The assay was evaluated by adding 0.1% trypan blue stain (TB) [the number of live (unstained) and dead (stained)] using a hemocytometer 64,65 . The parasite mortality (in %) for each bacterial supernatants sample was caluclated according to the formula: % Mortality of parasites = (Control Negative-Test sample) × 100%/Control negative. Only 100% inhibition of the parasite was considered when no motile parasite was observed.
Two negative and one positive control group were included in each experiment. Bacterial culture medium (TSB) and parasite medium was used as a negative control. Metronidazole (Specia Rhone Poulenc Rorer, Paris, France) for T. vaginalis and E. histolytica, Chlorhexidine (Sigma, Spain) for A. castellanii, N-methyl meglumine (Glucantime™, Rhone Poulenc, France) for L. tropica and Benzimidazole (Sigma, Spain) for T. cruzi were used as positive controls. Each assay was performed at least three times in triplicate.
Identification of antiprotozoal compounds using the easyPACId method. Generating promoter exchange mutant strains. The easyPACId approach method recently developed by Bode et al. 43 was used to identify the antiprotozoal compounds in Xenorhabdus spp. bacteria. Briefly, ∆hfq mutants of each bacterial species (X. budapestensis, X. cabanillasii, X. doucetiae, X. hominickii, X. nematophila, X. stockiae and X. szentirmaii) www.nature.com/scientificreports/ were first generated and then the native promoter regions of selected natural product biosynthetic gene clusters of these bacteria were exchanged with the chemically inducible promoter P BAD (addition of L-arabinose) via integration of the pCEP-KM plasmid 43,66 . This allows the selective production of a desired single natural product compound class and enables direct bioactivity analysis of the corresponding supernatant instead of timeconsuming isolation of single compound(s) from analytically complex wild type extracts. The generation of the described Xenorhabdus spp. ∆hfq as well as Xenorhabdus spp. ∆hfq pCEP-KM-xy mutants listed in Table 3 (xy describes the locus of the first biosynthetic gene cluster) is described in detail by Bode et al. 43 . Testing the antiprotozoal activity of cell-free supernatants of mutant strains. Antiprotozoal activity of 5-day-old cell-free supernatants of wild type strains, as well as induced (with arabinose) and noninduced (without arabinose) promoter exchange mutant strains were tested in microdilution bioassay as previously described in the in vitro antiprotozoal activity tests section.

Obtaining cell-free supernatants from different
Anti-protozoal activity of bioactive extracts obtained from hfq mutants. As a last step, extracts containing identified anti-protozoal compounds were tested again on the parasite species at different concentrations ranged from 10 to 0.078% (v/v). The same experimental method used in antiprotozoal activity tests was carried out here. Anti-protozoal bioactive compound extraction was performed by culturing induced X. nematophila Δhfq_ pCEP_ kan_XNC1_1711 for xenocoumacin production and X. doucetiae Δhfq_P BAD _ PAX_km for PAX peptide production in LB (6L) with 2% XAD® resin at 30 °C for 3 days. Afterwards the resin was exhaustively extracted with methanol (3 × 2 L) at 24 ± 1 °C and concentrated under reduced pressure to give a crude extract enriched by the desired natural compound class. The extracts were then dissolved in DMSO and prepared as a stock solution with distilled water. Fabclavine was obtained by concentrating the supernatant of the induced X. cabanillasii Δhfq_128-129 culture 10-fold using an evaporator.
Statistical analysis. Differences in antiprotozoal activity of the supernatants were compared with one-way ANOVA and the means separated using Tukey's test. P values < 0.05 were considered as significant 68 . The results are reported as mean ± SD for all values.
Ethics approval. This article does not contain any studies with human participants or animals performed by any of the authors.

Identification of antiprotozoal compounds. The promoter exchange mutants in Δhfq background
revealed that antiprotozoal bioactive compounds produced by Xenorhabdus bacteria were fabclavines, xenocoumacins, xenorhabdins and PAX peptides ( Table 3, Fig. 1). The supernatants obtained from induced mutants showed very high mortality against the parasite cells, non-induced mutants of the same compounds exhibited no activity (Table 3).
Fabclavines produced by X. cabanillasii, X. hominickii and X. stockiae species had antiprotozal activity against A. castellanaii, T. vaginalis L. tropica and T. cruzi parasites. Fabclavines produced by X. budapestensis was not effective against T. vaginalis. Xenorhabdus szentirmaii also produces fabclavines being only effective against A. castellanii and L. tropica with no antiprotozoal activity against T. vaginalis and T. cruzi.
Xenocoumacins produced by X. nematophila species was the bioactive antiprotozoal compound against all tested pathogens. In contrast to other species, X. doucetiae species produce more than one antiprotozoal compound. Δhfq_P BAD _PAX_km of X. doucetiae producing PAX peptides exhibited antiprotozoal effect on A. castellanii and T. vaginalis, but L. tropica was killed by xenocoumacins and xenorhabdins. Xenorhabdus doucetiae Δhfq_P BAD _xcnA_km showed antiprotozoal activity only with xenocoumacins against T. cruzi ( Table 3). The active compound in P. namnaoensis which was the only species that caused mortality on E. histolytica trophozoites was not identified due to the lack of promoter exchange mutants of this species.
Anti-protozoal activity of bioactive extracts obtained from hfq mutants. Supernatants  www.nature.com/scientificreports/ concentrations; no mortality was observed in the control (Fig. 2). Overall fabclavine molecules were highly effective on all tested parasite species even at very low concentrations.

Discussion
Our data revealed that Xenorhabdus and Photorhabdus produce antiprotozoal compounds effective on human pathogenic protozoa. However, not all Xenorhabdus or Photorhabdus species showed this activity. Except for E. histolytica, only some of Xenorhabdus species exhibited antiprotozoal activity. It was reported that Xenorhabdus bacteria produce broad-spectrum compounds with various activity against several organisms such as bacteria, fungi, insects, nematodes, mites, protozoa etc. to protect and bioconvert the host cadaver [69][70][71][72] . With the easy-PACId approach we were able to assign the described activities on respective natural products from Xenorhabdus. The bioactivity of fabclavines could be confirmed for X. budapestensis, X. cabanillasii, X. hominickii, X. stockiae and X. szentirmaii mutants. Biochemically fabclavines are peptide/polyketide hybrids connected to a polyamine moiety generated by a fatty acid/polyketide synthase with similarity to enzymes producing polyunsaturated fatty acids (PUFAs) 67,73,74 . Fabclavines 1a and 1b exhibit various bioactivities against different bacterial, fungal and protozoal organisms 73 and due to such broad-sprectrum activity, fabclavines might serve as protective agents against saprophytic food competitors/microorganisms that attack insect cadavers; this enables Xenorhabdus/Steinernema to maintain a monoculture in the infected insect 73 . Fabclavines are structurally very similar to zeamines identified in Serratia plymuthica so might similarly permeabilize artificial bacterial and eucaryotic model membranes 75,76 .
A structurally yet-undentified fabclavine derivative from X. innexi (Xlt) induces membrane degradation at low concentrations in selected mosquito cell lines which led to apoptosis 77 . Production of fabclavine is widespread in Xenorhabdus strains whereas, except for Photorhabdus asymbiotica, other Photorhabdus species do not produce fabclavines 42 . This can explain partially why none of our tested Photorhabdus species showed antiprotozoal activity. However, X. bovienii is a producer of only the polyamine part of fabclavine 74 and it did not exhibit any activity. There are 32 different types of fabclavine with important variations among their activity 67 .
Xenorhabdus nematophila and X. doucetiae species do not produce fabclavine 42 but they are effective species on tested parasites except for E. histolytica. According to promoter exchange data, it became obvious that X. nematophila and X. doucetiae perform this task with different compounds. Xenocoumacins are produced using  www.nature.com/scientificreports/ biosynthetic gene cluster were only be identified from seven Xenorhabdus subspecies (X. nematophila, X. indica, X. miraniensis, X. stockiae, X. kozodoii, X. mauleonii and X. doucetiae) 42 According to our data, we have determined that xenorhabdins and PAX peptides produced by X. doucetiae are other effective secondary metabolites. Xenorhabdins are dithiolopyrrolone compounds 82 and it is reported that they have antibacterial, antifungal, and insecticidal activities [83][84][85] . Their suggested mode of action is the inhibition of RNA synthesis affecting translation as similar to xenocoumacins 86,87 . PAX peptides are lysine-rich cyclolipopeptides. Gualtieri et al. 88 first described five PAX peptides from X. nematophila and then additional eight PAX peptides were identified, and their structures elucidated by Fuchs et al. 73 . Three NRPS genes (paxABC) are responsible for the biosynthesis of the PAX compounds. These peptides have antifungal and antibacterial activity. They exhibited strong anti-fungal activity against the opportunistic human pathogen Fusarium oxysporum as well as several plant pathogenic fungi 88 .
Interestingly, among the tested 27 Xenorhabdus and Photorhabdus strains only P. namnoensis appears to have acquired amoebicidal property which is effective on E. histolytica trophozoites. The bioactive compound responsible for this activity and its mode of action needs to be identified in the future.
The determined bioactive compounds may offer new opportunities for treating important parasitic diseases or be useful as lead compounds in the development of new antiprotozoal agents. For this purpose, new bioactive compounds should have no or very low cytotoxicity on human cells. Bode et al. 43 tested the efficacy of bioactive compounds isolated from Xenorhabdus and Photorhabdus bacteria on the human microvascular endothelial cell (EC) line (CDC.EU.HMEC-1). Fabclavine, PAX peptide, xenocoumacin and xenorhabdin had no or low impact on the metabolic activity, apoptosis and cell cycle G2-block. However, xenocoumacin and xenorhabdin exhibited toxic effects on cell proliferation.
In conclusion, this is the first extensive study screening the anti-protozoal activity of Xenorhabdus and Photorhabdus secondary metabolites against important human parasites A. castellanii, E. histolytica, T. vaginalis, L. tropica and T. cruzi and using the easyPACId technique to identify new potential antiprotozoal compounds. Future studies should investigate in detail the mode of action of these promising antiprotozoal compounds. Also, after a close structural investigation of these NPs, novel and safer pharmaceutical drugs can be potentially designed and synthesized.

Data availability
All data generated from this study are included in this article.